Project Summary/Abstract Vestibular disorders affect more than 90 million individuals in the United States, leading to challenges with ac- tivities of daily living and impaired overall quality of life. Although vestibular rehabilitation may help alleviate patients? symptoms, there are currently no biological treatments to reverse vestibular dysfunction. Whereas non-mammalian species such as chickens and zebrafish robustly regenerate the mechanosensory hair cells required for function after damage, the mammalian utricle (gravity-sensing organ) is only capable of limited re- generation. Atoh1, the transcription factor required for hair cell development, has previously been used to in- duce hair cell formation. However, the molecular features and functionality of the regenerated hair cells in the spontaneously and Atoh1-overexpressed regenerating utricles have not been clearly defined. Together, these gaps in knowledge create a critical bottleneck in our attempt to better understand the mechanisms of mature mammalian hair cell regeneration. The overall objective of this proposal is to examine the molecular features and function of the hair cell- synaptic complex in the spontaneously and Atoh1-enhanced regenerating adult mouse utricle. The hypothe- sis is that maturation of the hair cell-synaptic complex correlates with improvement of vestibular function, and that this regeneration can be driven further by Atoh1 overexpression. Completion of the following specific aims should test the central hypothesis and, thereby, attain the objective of this application. Specific Aim 1: Exam- ine molecular features of the spontaneously recovering hair cell-synaptic complex. Specific Aim 2: Restore vestibular function via spatiotemporal genetic manipulation of Atoh1. We will use a Cre-LoxP based transgenic approach to fate-map supporting cells after hair cell damage in mature mice. We will serially evaluate mice for vestibular function and examine utricles histologically up to six months after damage. We will then correlate improved histology with recovery of vestibular function. Successful execution of the work described in this proposal will result in a greater understanding of the mo- lecular features of the hair cell-synaptic complex in the mammalian inner ear. This contribution will be signifi- cant in two ways: first, it will establish an important but poorly understood relationship between hair cell regen- eration and functional recovery. Secondly, it will extend our understanding of the efficacy and limitation of using Atoh1 overexpression to stimulate hair cell regeneration. Such knowledge on inner ear hair cell regeneration in preclinical models will help construct the fundamental building blocks of future therapeutics to restore vestibu- lar function in humans.